The use of antimicrobial therapy to treat bacterial infections and diseases in humans and animals is well known throughout the world. Successful therapy requires selection of an antimicrobial agent that is specific to the bacteria causing the infection or disease. This antimicrobial agent selection is typically made by the health practitioner with the assistance and input of clinical microbiological laboratories which can measure the in vitro susceptibility of bacteria to antimicrobial agents by a variety of methods.
In many such laboratories a standard method of assessing the susceptibility of common pathogens to antimicrobial agents is by agar disk diffusion, commonly known as Disk Susceptibility testing, whereby a disk impregnated with an antimicrobial agent is pressed onto agar that has been infused with the suspected pathogen that has been isolated from a specimen taken from the patient, the agar is incubated and microbial growth or inhibition around the disk is observed and recorded. The National Committee for Clinical Laboratory Standards (NCCLS) has formulated specific uniform methods for such Disk Susceptibility testing, criteria for quality control testing and tables for quantitatively measuring the degree of pathogenic bacteria inhibition. See xe2x80x9cPerformance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Seventh Edition,xe2x80x9d NCCLS Bulletin M2-A7 (January 2000).
Many common pathogens are xe2x80x9cfastidiousxe2x80x9d microorganisms in the sense of requiring a special culture medium and a controlled environment to grow. The NCCLS Bulletin recommends the use of a Gonococcal (GC) agar medium supplemented by a 1% defined growth supplement for Disk Susceptibility testing of the fastidious microorganism N. gonnorrhoeae. See NCCLS Bulletin M2-A7 at page 10 and Jones et al., 27 J. Clin. Microbiology 2758 (1989). However such a supplemented GC agar medium was not recommended for use with any other fastidious microorganisms. The NCCLS Bulletin recommends that a variety of culture media having different formulations be used to test fastidious microorganisms such as Haemophilus species, N. gonorrhoeae, Streptococcus pneumoniae, Streptococcus viridans and xcex2-hemolytic Streptocci. Ibid, page 9. This requires the preparation of a different culture medium for each suspected pathogen, a cumbersome and time-consuming task when, as is usually the case, the identify of the pathogen is unknown.
There is therefore a need in the art for a single culture medium suitable for antimicrobial Disk Susceptibility testing for multiple fastidious microorganisms and even simultaneous testing for such microorganisms.
The foregoing need is met by the present invention, which provides a culture medium suitable for simultaneous antimicrobial Disk Susceptibility testing of multiple fastidious microorganisms, including Haemophilus species, Streptococcus pneumoniae, Streptococcus viridans species, xcex2-hemolytic Streptocci, Neisseria species and Campylobacter species.
The present invention lies in the discovery that the further supplementation of a particular known supplemented GC agar medium provides a culture medium useful in Disk Susceptibility testing for multiple fastidious pathogenic microorganisms.
The known supplemented GC agar medium (hereafter referred to as xe2x80x9cbase GC mediumxe2x80x9d) comprises a mixture of deionized water, agar, peptones, beef and yeast extracts, starch, glucose and phosphate buffer components. More specifically the non-aqueous portion of this base GC medium comprises the components noted below present in the amounts noted:
The foregoing non-aqueous portion is preferably mixed with deionized water in a concentration of about 40 g/L of water.
The deionized water may have a pH of 5 to 7, preferably 7, since the final pH of the culture medium of the present invention is preferably 7.3xc2x10.1 at ambient temperature; its bacteriological purity should be less than 100 CFU/mL with a TOC content of less than about 712 ppb and a resistivity of xe2x89xa72 megohms.cm at 25xc2x0 C. The agar used in the base GC medium is preferably of the Iber type derived from Gelidium seaweeds that is of pharmaceutical grade, commercially available from DMV International Nutritionals of Fraser, N.Y. (hereafter xe2x80x9cDMVxe2x80x9d).
The remaining components of the base GC medium and their commercial availability is as follows: casein peptone, a pancreatic digest of the complex of milk proteins known by that name, available as Product No. CE90-M from DMV; meat peptone, an enzymatic digest of animal tissue that is preferably a 50/50 mixture (w/w) of Product No. 102, available from Global and Product No. AE80M, available from DMV; proteose peptone, available as Product No. PP90M from DMV; peptonized milk, available as Product No. 5X59048 from Quest International Co. of Norwich, New York; beef extract, available in powder form as Product No. 150 from Global BioIngredients of Tampa, Fla. (hereafter xe2x80x9cGlobalxe2x80x9d); yeast extract, available as Product No. 151 from Global; starch (preferably reagent grade potato starch) available as Product No. S-123 from Pfanstiehl Laboratories, Inc. of Chicago, Ill.; and glucose, available as Product No. DE140 from Spectrum Laboratory Products, Inc. of Gardena, Calif. (hereafter xe2x80x9cSpectrumxe2x80x9d). The remaining chemical components of NaCl, K2HPO4, KH2PO4 and Na2CO3 are readily commercially available from many sources, and are preferably of ACS reagent grade.
Further supplementation of the foregoing base GC medium with a multi-component amino acid supplement and with laked horse blood allows its use for antimicrobial Disk Susceptibility testing for at least six classes of fastidious microorganisms, including Neisseria species, Haemophilus species, Streptococcus pneumoniae, Streptococcus viridans species, xcex2-hemolytic Streptocci and Campylobacter species.
The amino acid component comprises deionized water and L-cysteine HCl, L-cystine, L-glutamine, guanine HCl, thiamine HCl, p-aminobenzoic acid (PABA), vitamin B12, cocarboxylase, nicotinamide adenine dinucleotide (NAD); adenine, glucose, HCl and Fe(NO3)3.9H2O (ferric nitrate nonahydrate).
In a preferred formulation the amino acid component comprises an aqueous solution of the following components in the following approximate concentrations
The non-aqueous components of the above amino acid component are commercially available as follows.
The amino acid component is preferably prepared by adding the L-cysteine HCl and L-cystine components to the deionized water in powdered form and stirring while adding the 12N HCl until the two powdered components are dissolved. The guanine HCl is added to the resulting solution and stirred for about 30 minutes until the same is dissolved. The remaining amino acid component ingredients are then added and the solution stirred until all components are in solution. The solution is then filtered with a 0.2 micron filter into a container that has been autoclaved at 127xc2x0 C. for at least 30 minutes.
The laked horse blood component preferably comprises lysed red blood cells extracted from defibrinated horse blood. A preferred method of lysing and extraction is to conduct three freeze/thaw cycles on the blood, followed by centrifuging at about 1200 rpm for about 20 minutes so as to separate white blood cells and red blood cell wall fragments. Following centrifugation, the resulting laked horse blood component is preferably filtered and added to the amino acid component-supplemented base GC medium in an amount sufficient to achieve a concentration of about 30 mL/L of water present in the base GC medium.
The base GC medium is preferably formulated by preparing the non-aqueous components in a dry powder form by first combining the agar, casein peptone, starch, glucose, peptonized milk and the chemicals in, for example, a Fitzmill grinder, then placing this mixture into a blender. The meat peptone, proteose peptone, beef extract and yeast extract components are then placed in the same blender and the entire mixture is blended for approximately two hours. The deionized water is then added and thoroughly mixed in a vessel equipped with a magnetic stirrer, pH adjusted to about 7.3 by the addition of Na2CO3, followed by autoclaving at 127xc2x0 C. and 22 atm for 20 to 55 minutes, depending upon batch size. The so-prepared base GC medium is then cooled to about 55xc2x0 C. and the amino acid component is added and thoroughly mixed, while allowing the mixture to further cool to about 48xc2x0 C. The laked horse blood component is filtered and mixed in with the base GC medium/amino acid component mixture as the same is poured into a sterile Petri dish; filtration is preferably with a 0.2 micron filter no more than 15 minutes prior to the mixing.